Annexins are a group of calcium-dependent, phospholipid-binding proteins. The calcium and phospholipid binding sites of most annexins are located in four repeated and highly conserved regions each of which contains about 70 amino acids. These proteins are widely distributed and at least nine members of the annexin family of proteins have been identified in mammalian tissues.
The lung is rich in annexins. Several members of the annexin family of proteins with apparent molecular weights ranging from about 32 to about 40 kDa have been isolated from lungs of animals. Annexin I, a 36 kD phospholipid binding protein, 36 kDa(PLBP), appears to be the most abundant of the annexin family of proteins. It is present in the lung in greater amounts than the related Annexin VIII, a 33 kDa phospholipid-binding protein, also known as 33 kDa(PLBP).
Compared to the whole lung, the alveolar epithelial type II cells in which the pulmonary surfactant complex is synthesized, stored and secreted have higher expression levels of Annexin I. In addition to the intracellular localization of Annexin I in alveolar type II cells, this protein has been found in lung lavage fluids from human and animals. A likely source of the Annexin I in the lung lavage fluid is the alveolar type II cells.
Using an antibody to Annexin I, researchers have found Annexin I and a smaller protein in the bronchoalveolar lavage (BAL) fluid of patients with lung diseases. The smaller protein was found to be a proteolytic breakdown product resulting from the action of neutrophil elastase upon Annexin I in the patients' BAL fluid. The discovery of the Annexin I breakdown product in human BAL fluid samples is consistent with the report that the Annexin I N-terminal region is subject to cleavage by various proteases to yield breakdown products.
I have discovered that a breakdown product of Annexin I, which has a molecular weight of about 33 kDa (33 kDa(BP)), immunoreacts with anti-Annexin I antibodies. I also have discovered that 33 kDa(BP) is cytotoxic and that it is present in higher concentrations in the BAL fluid of patients with lung diseases, including cystic fibrosis (CF), and premature infants with chronic lung disease, bronchopulmonary dysplasia, (BPD) than in normal humans. In addition, the BAL fluid of patients with lung disease was found to contain less Annexin I than that of normal humans.
Based on these and other discoveries, I have found that administering Annexin I or Annexin VIII, to patients with lung disease can be beneficial to such patients. I also have made the further discovery that the administration of Annexin I or Annexin VIII, can be beneficial in the treatment of endotoxin toxicity and inflammation. The major pathogenesis of endotoxin toxicity leads to inflammation and septic shock.
Bacteria or bacterial products, such as endotoxin from gram-negative bacteria, activate host responses during infectious and inflammatory processes. Endotoxin, also known as lipopolysaccharide (LPS) for its chemical structure consisting of a polysaccharide part and a hydrophobic lipid part, can induce a wide variety of different types of cells including macrophages, polymorphonuclear leukocytes, and endothelial cells to release a number of inflammatory mediators, such as prostaglandins or cytokines. In localized infections, endotoxin is largely restricted to inflammatory sites, enhancing host defense. However, if the infection is not brought under control, endotoxin and/or inflammatory mediators may reach the circulation, predisposing the microvasculature to thrombosis and can lead to systemic endotoxemia or sepsis and associated complications including septic shock, adult respiratory distress syndrome, and multiorgan failure.
Septic or endotoxin shock is an acute and serious cardiovascular collapse resulting from the systemic response to a bacterial infection. It is manifested by hypotension, a reduced response to vasoconstrictors, generalized tissue damage and multi-organ failure. It is the most common cause of death in the intensive-care unit; there are about 400,000 cases of septicemia per year in the United States with mortality rates between 25% and 50%. The steadily increasing incidence of septic shock stems from an increasing proportion of elderly in the population, increasing frequency of invasive surgical procedures, extensive use of immunosuppressive and chemotherapeutic agents, and increasing prevalence of chronic debilitating conditions. Because the mechanisms underlying sepsis and septic shock are not yet known, therapeutic interventions have been largely ineffective. At present, there is no effective treatment for septic shock.
Phospholipase A.sub.2 (PLA.sub.2) is an enzyme which plays a role in a pathway which can lead to inflammation. Inflammation in the lung is caused by inflammatory mediators released by cells, including histamine, cytotoxins (tumor necrosis factor alpha, interleukin-1, etc), and leukotrienes. These mediators cause blood vessels to leak and lung tissues to swell, and act to contract the smooth muscles of the airways to cause bronchial constriction. The inflammation response, originally designed to combat threats, is in itself the mediator of substantial tissue damage. Patients with conditions such as asthma, cystic fibrosis, and bronchopulmonary dysplasia experience chronic inflammation of the lung.
Phospholipase A.sub.2 is an important enzyme in a synthetic pathway which can lead to the formation of arachidonic acid. Arachidonic acid in turn may be acted upon by cyclooxygenase to form prostaglandins, by lipooxygenase to form leukotrienes, and by monooxygenase to form epoxides all which can cause inflammation.
Compounds that interfere with this pathway can be useful in preventing inflammation. For example, prostaglandin inhibitors are widely known and used for inflammation, and a lipooxygenase inhibitor to prevent formation of leukotrienes has recently come on the market for treatment of asthma.
It would be advantageous to have methods of inhibiting PLA.sub.2 activity and suppressing the activity of PLA.sub.2 simulators, thereby preventing or treating inflammation, especially that which accompanies lung diseases, including cystic fibrosis (CF) and bronchopulmonary dysplasia (BPD). It also would be advantageous to have a method of evaluating agents that might be effective to suppress the activity of PLA.sub.2 simulators in a patient's body fluid.